Biocatalysis has become an important technology in the field of biosynthesis because of its mild reaction conditions, high efficiency, high specificity and low price. There are a series of highly integrated metabolic networks in the biosynthesis system, and the study of multi-enzyme catalytic system has become an inevitable trend in the field of biosynthesis, so it is of great significance to explore the unknown multi-enzyme synthesis path based on the known products. In this review, the concepts of multi-enzyme system and retrosynthesis process are introduced. And the design methods, advantages and disadvantages of retrosynthesis tools are summarized. Then the tools are divided into host-based and host-less tools. For each of these two types, some representative retrosynthesis tools are listed to analyze their respective design processes and differences. Finally, the possibility of AI-assisted multi-enzyme system is discussed and the optimization and development of multi-enzyme pathway construction tools are forecasted.